2-(3,8-dioxatricyclo[5.1.0.0 ]oct-5-en-5-yl)-4h-pyran-4-one

ABSTRACT

This disclosure describes 2-(3,8-dioxatricyclo-(5.1.0.02,4)oct5-en-5-yl)-4H-pyran-4-one, a new compound which exhibits antimicrobial activity. This new compound is formed during the cultivation under controlled aerobic conditions of an undetermined filamentous fungal species NRRL 3938.

ie States Patent Earbatschi et al.

[ 51March 13, 1973 2-(3,8-DIOXATRICYCLO[5.1.0.0 2,4 ]OCT-S-EN-S-YL)-4H-PYRAN-4-ONE Inventors: Ferdinand Barbatschi, Montvale,

N.J.; Donald Bruce Borders, Suffern; Anthony Joseph Shay, Pearl River, both of N.Y.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: May 21, 9171 Appl. No.: 145,968

US. Cl. ..260/345.9, 424/283, 195/81 Int. Cl. ..C07d 7/18 Field of Search ..260/245.9

Primary ExaminerNorma S. Milestone Attorney-Edward A. Conroy, Jr.

[57] ABSTRACT 1 Claim, No Drawings No other spore types produced.

. Cultures on potato dextrose agar spreading rapidly,

2-(3,8-DIOXATRICYCLO[5.1.0.0 ]CT-5-EN-5- Cultures on Czapeks solution agar effuse, covering YL)-4ll-PYRAN-4-ONE the Petri dish in 14 days. Growth colorless, very thin,

reverse also colorless. Occasional chlamydospores formed. No other spore types observed. This invention relates to a new antibiotic, 2-(3,8- 5 Cultures com mealagar effusmspreading p y,

BRIEF SUMMARY OF THE INVENTION dioxatric l0[5 l 0 t-5- .5- ])-4H- 4- covering the Petri dish in 14 days. Growth whitish, very one, which may be represented by the following structhin, reverse whitish amy pores pr duced tural formul sparingly. No other spore types produced.

It is to be understood that for the production of the new antibiotic, 2-(3,8-dioxatricyclo[5.l .0.0]oct-5- en-S-yl)--4H-pyran-4-one, the present invention is not limited to this particular organism only, nor to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. In fact, it is desired and intended to include the use of mutants produced from the described organism by various means such as X-radiation, ultraviolet radiation, nitrogen mustard, phage exposure, and the like.

The invention includes within its scope the antibiotic THE FERMENTATION PROCESS compound in dilute forms, as crude concentrates, and

in pure crystalline form as well as methods of preparing Cultivation of the organism Z1220 may be carried the antibiotic. This novel compound possesses broadout in a wide variety of liquid culture media. Media spectrum antibiotic activity in vitro against Gram-negawhich are useful for the production of the novel antive and Gram-positive bacteria. tibiotic include an assimilable source of carbon such as starch, sugar, molasses, glycerol etc., an assimilable DETAILED DESCRIPTION OF THE INVENTION source of nitrogen such as protein, protein hydrolysate, The new compound 2-(3,8-dioxatricyclo[5.l.0.0 P yp p amino acids com steep q em and 5 5 4 4 is f d during the p inorganic anions and cations, such as potassium, soditivation under controlled aerobic conditions of an un um, calcium, sulfate Phosphaie, chloride, Trace determined filamentous fungal species which we have elemems such as boron, molybdenum, pp are denominated culture Z1220. This new antibioticpp as impurities of other constituents of the producing fungal species was isolated from a soil sammedla- Aeration ls Provided y forcing sterile air A viable culture f the organism has been through or onto the surface of the fermenting medium. deposited with the Culture Collection Laboratory, Agitation is Provided by a mechanical lmpeller- Northern Utilization Research and Development Divifoaming agent such as 1 Percent Octadecanol in lard sion, U.S. Department of Agriculture, Peoria, 111. and ollmay be added as neededhas been added to its permanent collection under its INOCULUM PREPARATION accession number NRRL 3938.

lnoculum is prepared by inoculating portions of DESCRIPTION OF THEORGANISM sterile liquid medium with scrapings or washings of culture Z1220 was grown on several diagnostic spores from an agar slant ofculture Z1220.The followmedia which normally support good growth and sporulng medium may be used: lation of fungal cultures. Culture Z1220 exhibited good Soybean meal 10 gm/liter mycehal development, but failed to produce spore Glucose 20 gmlmer types useful for taxonomic purposes. It did, however, (30", leep liquor 5 In r mproduce abundant chlamydospores, but these are of lit- Calclum calbmm 3 s Water sto 1 ft tle diagnostlc value since they are commonly produced q by a diversity of organisms. In the absence of suitable taxonomic criteria for finite identification, the organism will have to remain an undetermined chlamydospore-producing fungal species. Following is a detailed description of culture Z1220:

Cultures on malt extract agar spreading rapidly, covering the Petri dish in 14 days. Growth grayishwhite, thin, heaviest in central zones; reverse yellowish. Mycelium septate, 3-4u in diameter, becoming thicker TANK FERMENTATION (6-8;.ta) with age. Chlamydospores produced abundantly both terminally and intercallary, mostly globose, ranging from 6-24p. in diameter, averaging about 15,12.

The inoculated medium is incubated at 24-30 C.

under aeration for 4872 hours. Two hundred milliliter portions are used to inoculate 12-liter batches of the same medium in a 20-liter glass fermentor. The inoculum mash is aerated with sterile air while growth is continued for 4872 hours. This in turn is used to inoculate a tank fermentor.

The inoculum, prepared as described above, is used to seed the following fermentation medium in tank fermentors:

. C l 20 covering the Petri dish in 14 days. Growth yellowish- 7:5 asses Emmet 10 gmlliter white, thin, becoming strongly zonate in wide bands. W fl r ;0 git/(liter cm s eep iquor gm 1 er Reverse yellowlsh-brown. Chlamydospores formed as Calcium carbonate 3 gmlmer on malt extract agar. No other spore types produced. Water qs to 1 liter Each tank is inoculated with 3 percent to percent of inoculum prepared as described above. Aeration is supplied at the rate of 0.5-1 .0 liter of sterile air per liter of mash per minute and the fermenting mixture is agitated by an impeller driven at 200-400 rpm. The temperature is maintained at 25-30 C., usually at 28 C. The fermentation is continued for 120-160 hours and then harvested.

ISOLATION PROCEDURE The harvested mash is filtered and the filtrate is adjusted to pH 7.0 with dilute sodium hydroxide and extracted with chloroform. The resulting extract is concentrated to dryness under reduced pressure. The residual mass is dissolved in chloroform. The solution is mixed with acetone and chilled. The crystalline product which separates is filtered off, washed with acetone, and dried in vacuo at room temperature.

ANTIMICROBIAL CHARACTERISTICS The novel compound of the present invention is useful as an antimicrobial agent and possesses broad spectrum activity in vitro against Gram-negative and Grampositive bacteria. This activity is determined against a variety of standard laboratory microorganisms as determined by the agar-dilution technique. In this assay, the compound to be tested is dissolved in dimethylsulfoxide so that 10.0 mg. of test compound is contained per milliliter of solution. Observing sterile techniques, tenfold serial dilutions are made of the test solution. Twotenths ml., 0.1 ml. and 0.05 ml. amounts of the original solution and of each of the decimal dilutions are then added to and mixed with ml. of warm sterile asparagine-meat extract agar capable of supporting growth of the test cultures. The standard sterile nutrient agar solutions containing the different dilutions of the test compound, along with suitable and comparable control dilutions containing no test compound, are then allowed to cool in Petri dishes thereby forming solid agar plates. The test organisms are prepared for use by growing on Trypticase Soy agar overnight. The organisms are harvested from mature agar slant cultures and are suspended in sterile physiological saline solution. Using the Steers Replicator, a standardized amount of the resulting live suspensions is then, still employing sterile techniques, imprinted upon the surfaces of each of the agar plates and the resulting inoculated plates are then incubated. After an appropriate period of time, each of the inoculated areas on each of the plates is inspected visually and the extent, if any, of growth is noted. The minimal inhibitory concentration (meg/ml.) is defined as the concentration of test compound causing complete inhibition of any particular organism.

In a representative operation, the minimal inhibitory concentrations of the compound of this invention against standard laboratory microorganisms, as determined in the above-described assay are set forth in Table 1 below:

TABLEI Minimal Inhibitory Concentration s/ ORGANISM The high in vitro antibacterial activity of the novel compound of the present invention makes it useful as an additive to materials which are subject to microbial deterioration such as cutting oils and fuel oils. It is also useful in soaps, shampoos, and topical compositions for the treatment of wounds and burns.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Inoculum Preparation A typical medium used to grow the inoculum was prepared according to the following formula:

Soy flour X 200 10 gmlliter Glucose 20 gm/liter Corn steep liquor 5 gm/liter Calcium carbonate 3 gm/liter Water qs to 1 liter Scrapings from an agar slant of culture Z1220 were used to inoculate two 500 ml. flasks each containing milliliters of the above sterile medium. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28 C. The resulting flask inoculum was transferred to a 20 liter glass fermentor containing 12 liters of the above sterile medium. The glass fermentor was aerated with sterile air while growth was carried out for 48 hours at 28 C. after which the contents were used to seed a 300 liter tank fermentor.

EXAMPLE 2 Fermentation Twelve liters of inoculum prepared as described in Example 1 was used to seed 300 liters of medium of the following formulation:

Cane molasses 20 gm/liter Glucose 10 gm/liter Soy flour (X 200) 10 gmlliter Com steep liquor 5 gm/liter Calcium carbonate 3 gm/liter Water qs to 1 liter The fermentation was carried out at 28 C. for

hours. Aeration was supplied at the rate of 0.5 liters of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 300 revolutions per minute.

5 6 EXAMPLE 3 U.\/. M 269 nm (6 16,800); [04 123 (C I 0.591 CHVl Isolation we claim: I The 300 liters of harvested mash was filtered. The fill. The compound 2-(3,8-dioxatricyclo[5. 1 0.0

trate was adjusted to pH 7.0 with dilute sodium hydrox 5 oct-5-en-5-yl)-4H-pyran-4-one represented by the foride and extracted with 150 liters of chloroform. The mula: resulting extract was concentrated to dryness under vacuum at a temperature below 40 C. The residual mass was dissolved in about 1 liter of chloroform. The

solution was filtered, mixed with 2 liters of acetone and maintained at 4C. for hours. The 58 gm. of crystalline product which separated was filtered off, washed with a small volume of acetone, and dried in vacuo at room temperature. The product was isolated as colorless needles, melting point 148 C. (decomposition); 

